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Abstract:

Disclosed is a method for treating a human having a disease associated
with leukocyte infiltration of mucosal tissues, comprising administering
to said human an effective amount of a human or humanized immunoglobulin
or antigen-binding fragment thereof having binding specificity for
α4β7 integrin. Preferably, no more than about 8 mg
immunoglobulin or fragment per kg body weight are administered during a
period of about one month.

Claims:

1. A method for inhibiting relapse and/or recurrence of quiescent
inflammatory bowel disease in a human, comprising administering to said
human an effective amount of a humanized immunoglobulin or
antigen-binding fragment thereof having binding specificity for human
α4.beta.7 integrin, said humanized immunoglobulin or
antigen-binding fragment comprising an antigen binding region of nonhuman
origin and at least a portion of an immunoglobulin of human origin,
wherein said humanized immunoglobulin or antigen-binding fragment is
administered in doses and the minimum interval between doses is a period
of about one month, and wherein no more than about 8 mg humanized
immunoglobulin or antigen-binding fragment per kg body weight are
administered during a period of about one month; wherein said humanized
immunoglobulin or antigen-binding fragment has binding specificity for
the α4.beta.7 complex, wherein said antigen-binding region
comprises three complementarity determining regions (CDR1, CDR2 and CDR3)
of a light chain variable region and three complementarity determining
regions (CDR1, CDR2 and CDR3) of a heavy chain variable region of the
amino acid sequence set forth below:
TABLE-US-00041
light chain: CDR1 SEQ ID NO: 9
CDR2 SEQ ID NO: 10
CDR3 SEQ ID NO: 11
heavy chain: CDR1 SEQ ID NO: 12
CDR2 SEQ ID NO: 13
CDR3 SEQ ID NO: 14.

10. The method of claim 1, wherein no more than 7 mg humanized
immunoglobulin or antigen-binding fragment per kg body weight are
administered during a period of about one month.

11. The method of claim 1, wherein each of said doses independently
comprise about 0.1 to about 8 mg humanized immunoglobulin or
antigen-binding fragment per kg body weight.

12. The method of claim 1, wherein each of said doses independently
comprises about 0.1 to about 5 mg humanized immunoglobulin or
antigen-binding fragment per kg body weight

13. The method of claim 1, wherein the interval between additional
subsequent doses is at least about 40 days.

14. The method of claim 1, wherein the interval between additional
subsequent doses is at least about 50 days.

15. The method of claim 1, further comprising administering an effective
amount of one or more additional therapeutic agents.

16. The method of claim 15, wherein the one or more additional
therapeutic agents is selected from the group consisting of steroids,
immunosuppressive agents, non-steroidal anti-inflammatory agents and
immunomodulators.

17. A method for inhibiting relapse and/or recurrence of quiescent
inflammatory bowel disease in a human, comprising administering to said
human an effective amount of a humanized immunoglobulin or
antigen-binding fragment thereof having binding specificity for human
α4.beta.7 integrin, said humanized immunoglobulin or
antigen-binding fragment comprising an antigen binding region of nonhuman
origin and at least a portion of an antibody of human origin, wherein
said humanized immunoglobulin or antigen-binding fragment is administered
in doses and the minimum interval between doses is a period of about one
month, and wherein no more than about 8 mg humanized immunoglobulin or
antigen-binding fragment per kg body weight are administered during a
period of about one month, wherein said humanized immunoglobulin or
antigen-binding fragment has binding specificity for the α4.beta.7
complex, and wherein said humanized immunoglobulin or antigen-binding
fragment thereof comprises the heavy chain variable region of SEQ ID NO:6
and the light chain variable region of SEQ ID NO:8.

18. The method of claim 17, wherein the interval between additional
subsequent doses is at least about 50 days.

19. The method of claim 17, wherein no more than 7 mg humanized
immunoglobulin or antigen-binding fragment per kg body weight are
administered during a period of about one month.

20. The method of claim 17, wherein each of said doses independently
comprise about 0.1 to about 8 mg humanized immunoglobulin or
antigen-binding fragment per kg body weight.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.
09/748,960, filed Dec. 27, 2000, which is a continuation of application
Ser. No. 09/550,082, filed Apr. 14, 2000. The entire teachings of the
above applications are incorporated herein by reference.

[0003] Inflammatory bowel disease (IBD), such as ulcerative colitis and
Crohn's disease, for example, can be a debilitating and progressive
disease involving inflammation of the gastrointestinal tract. Affecting
an estimated two million people in the United States alone, symptoms
include abdominal pain, cramping, diarrhea and rectal bleeding. IBD
treatments have included anti-inflammatory drugs (such as,
corticosteroids and sulfasalazine), immunosuppressive drugs (such as,
6-mercaptopurine, cyclosporine and azathioprine) and surgery (such as,
colectomy). Podolsky, New Engl. J. Med., 325:928-937 (1991) and Podolsky,
New Engl. J. Med., 325:1008-1016 (1991). However, such therapeutic agents
have not been effective in maintaining remission of IBD.

[0004] Antibodies against human α4β7 integrin, such as murine
monoclonal antibody (mAb Act-1), interfere with α4β7 integrin
binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) present
on high endothelial venules in mucosal lymph nodes. Act-1 was originally
isolated by Lazarovits, A. I., et al., J. Immunol. 133:1857-1862 (1984),
from mice immunized with human tetanus toxoid-specific T lymphocytes and
was reported to be a mouse IgG1/κ antibody. More recent analysis of
the antibody by Schweighoffer, T., et al., J. Immunol. 151:717-729 (1993)
demonstrated that it can bind to a subset of human memory CD4+ T
lymphocytes which selectively express the α4β7 integrin.
However, a serious problem with using murine antibodies for therapeutic
applications in humans is that they are highly immunogenic in humans and
quickly induce a human anti-murine antibody response (HAMA), which
reduces the efficacy of the mouse antibody in patients and can prevent
continued administration. The HAMA response results in rapid clearance of
the mouse antibody, severely limiting any therapeutic benefit.

[0005] Thus, a need exists for improved therapeutic approaches to
inflammatory bowel diseases and other inflammatory disorders of mucosal
tissues.

SUMMARY OF THE INVENTION

[0006] The invention relates to a method of administering an antibody
(e.g., humanized antibody, human antibody). In one aspect the invention
is a method of treating a human having a disease associated with
leukocyte infiltration of mucosal tissues comprising administering to the
human an effective amount of an immunoglobulin having binding specificity
for α4β7 integrin. In preferred embodiments no more than about
8 mg immunoglobulin per kg body weight is administered in a period of
about one month. In particular embodiments, the immunoglobulin can
include one or more complementarity determining regions (CDRs) having the
amino acid sequence of a CDR of murine Act-1 mAb. LDP-02 is a preferred
antibody for administration. The immunoglobulin can be administered in
multiple doses and the interval between doses can be at least 1 day or
longer. In particular embodiments, the interval between doses can be at
least about 7, 14 or 21 days or about one month. In one embodiment, the
amount of immunoglobulin administered per dose can be an amount which is
sufficient to achieve about 50% or greater saturation of α4β7
binding sites on circulating lymphocytes and/or about 50% or greater
inhibition of α4β7 integrin expression on the surface of
circulating lymphocytes for a period of at least about 10 days following
administration of the dose. In another embodiment, the amount of
immunoglobulin administered per dose can be an amount which is sufficient
to achieve and maintain a serum concentration of said immunoglobulin of
at least about 1 μg/mL for a period of about 10 days following
administration of the dose.

[0007] The immunoglobulin can be administered alone or together with one
or more other agents to treat a disease associated with leukocyte
infiltration of mucosal tissues. For example, the immunoglobulin can be
administered with steroids, immunosuppressive agents, non-steroidal
anti-inflammatory agents or immunomodulators. In a preferred embodiment
immunoglobulin is administered to treat a human having an inflammatory
bowel disease, such as Crohn's disease or ulcerative colitis.

[0009] FIG. 2 is an illustration of the nucleotide sequence of a double
stranded nucleic acid (coding strand, SEQ ID NO:3; non-coding strand, SEQ
ID NO:16) encoding the mouse Act-1 antibody heavy chain variable region
and signal peptide, and the deduced amino acid sequence of the Act-1
heavy chain variable region and heavy chain signal peptide sequence (SEQ
ID NO:3). The nucleotide sequence of the variable region is joined to a
nucleotide sequence which encodes a deduced mouse Act-1 heavy chain
signal peptide sequence, to yield a composite sequence. (The identity of
the primer which amplified the heavy chain region was deduced from the
degenerate sequence, and an amino acid sequence for the signal peptide
was derived from the primer, downstream sequence and sequences of other
signal peptides. The signal peptide shown may not be identical to that of
the Act-1 hybridoma.)

[0010] FIG. 3 is an illustration of the nucleotide sequence (SEQ ID NO:5)
and amino acid sequence (SEQ ID NO:6) of a portion of the heavy chain of
a humanized Act-1 antibody (LDP-02) with a heavy chain signal peptide.

[0011] FIG. 4 is an illustration of the nucleotide sequence (SEQ ID NO:7)
and amino acid sequence (SEQ ID NO:8) of a portion of the light chain of
a humanized Act-1 antibody (LDP-02) with a light chain signal peptide.

[0013] FIG. 6 is a graph showing mean serum LDP-02 levels (μg/ml) in
healthy men over time following a single administration of LDP-02. Mean
serum LDP-02 levels became negligible by day 36 following administration
of 0.15 mg/kg by intravenous (IV) (-.diamond-solid.-) or subcutaneous
(SC) (-.box-solid.-) injection and following administration of 0.5 mg/kg
by intravenous injection (-.tangle-solidup.-). However serum LDP-02 was
still measurable beyond day 36 following administration of 1.5 mg/kg
(-x-) or 15 mg/kg (-*-) by intravenous injection.

[0014] FIG. 7 is a graph showing persistent loss of α4β7 signal
(detected with Act-1 mAb) following administration of LDP-02. About 90%
of α4β7 signal was rapidly lost (MESF=10%) after
administration of LDP-02 and persisted following administration of all
LDP-02 doses. Between about day 7 and day 22, α4β7 signal
started to return to baseline for the 0.15 mg/kg IV dose group
(-.diamond-solid.-) and for the 0.15 mg/kg SC dose group (-.box-solid.-).
Between day 22 and day 36, α4β7 signal started to return to
baseline for the 0.5 mg/kg IV (-.tangle-solidup.-) dose group. At the
higher doses of LDP-02 studied (1.5 mg/kg (-x-) and 2.5 mg/kg (-*-)),
loss of α4β7 signal persisted for longer than 36 days
following single IV doses. For the 2.5 mg/kg dose group (-*-), loss of
α4β7 signal persisted up to about Day 70 (data provided in
Appendix to Study L297-007). MESF: mean equivalent soluble fluorescence.

[0015] FIG. 8 is a graph showing mean serum LDP-02 levels (μg/ml) in
patients with ulcerative colitis over time following a single
administration of LDP-02. Mean serum LDP-02 levels rose rapidly following
administration of LDP-02. The concentration of serum LDP-02 fell to below
1.0 μg/mL in patients administered LDP-02 at 0.15 mg/kg by intravenous
(-.tangle-solidup.-) or subcutanious (- -) injection by 10 days following
dosing. However, serum LDP-02 concentrations remained above 1.0 μg/mL
for about 20 days following administration of 0.5 mg/kg by intravenous
injection (-.box-solid.-). The serum concentration of LDP-02 remained
above 1 μg/mL for about 60 days following administration of 2.0 mg/kg
by intravenous injection (--).

[0016] FIG. 9 is a graph showing persistent loss of α4β7 signal
(detected with Act-1 mAb) following administration of LDP-02. About 90%
of α4β7 signal was rapidly lost (MESF=10%) after
administration of LDP-02 and the duration of signal loss was dependent
upon dose. Starting at about Day 10, α4β7 signal started to
return to baseline for the group administered 0.15 mg/kg of LDP-02 by IV
(-.box-solid.-) or SC (-.diamond-solid.-) injection. However,
α4β7 signal started to return to baseline between day 30 and
day 60 for the group administered 0.5 mg/kg (-.tangle-solidup.-)
intravenously, and after day 60 for the group administered 2.0 mg/kg
(-x-) intravenously (data provided in Appendix to Study L297-006). MESF:
mean equivalent soluble fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to a method of administering an
antibody (immunoglobulin) to a subject. In one aspect, the antibody to be
administered is a human or humanized antibody having binding specificity
for α4β7 integrin (e.g., mammalian α4β7 (e.g.,
human (Homo sapiens) α4β7). Preferably, the human or humanized
immunoglobulins can bind α4β7 integrin with an affinity of at
least about 107 M-1, preferably at least about 108
M-1, and more preferably at least about 109 M-1. In one
embodiment, the humanized immunoglobulin includes an antigen binding
region of nonhuman origin which binds α4β7 integrin and a
constant region derived from a human constant region. In another
embodiment, the humanized immunoglobulin which binds α4β7
integrin comprises a complementarity determining region of nonhuman
origin and a variable framework region of human origin, and if desired, a
constant region of human origin. For example, the humanized
immunoglobulin can comprise a heavy chain and a light chain, wherein the
light chain comprises a complementarity determining region derived from
an antibody of nonhuman origin which binds α4≈7 integrin
and a framework region derived from a light chain of human origin, and
the heavy chain comprises a complementarity determining region derived
from an antibody of nonhuman origin which binds α4β7 integrin
and a framework region derived from a heavy chain of human origin.

[0018] Naturally occurring immunoglobulins have a common core structure in
which two identical light chains (about 24 kD) and two identical heavy
chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of
each chain is known as the variable (V) region and can be distinguished
from the more conserved constant (C) regions of the remainder of each
chain. Within the variable region of the light chain is a C-terminal
portion known as the J region. Within the variable region of the heavy
chain, there is a D region in addition to the J region. Most of the amino
acid sequence variation in immunoglobulins is confined to three separate
locations in the V regions known as hypervariable regions or
complementarity determining regions (CDRs) which are directly involved in
antigen binding. Proceeding from the amino-terminus, these regions are
designated CDR1, CDR2 and CDR3, respectively. The CDRs are held in place
by more conserved framework regions (FRs). Proceeding from the
amino-terminus, these regions are designated FR1, FR2, FR3, and FR4,
respectively. The locations of CDR and FR regions and a numbering system
have been defined by Kabat et al. (Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of
Health and Human Services, U.S. Government Printing Office (1991)).

[0020] The term "immunoglobulin" as used herein includes whole antibodies
and biologically functional fragments thereof. Such biologically
functional fragments retain at least one antigen binding function of a
corresponding full-length antibody (e.g., specificity for α4β7
of Act-1 antibody), and preferably, retain the ability to inhibit the
interaction of α4β7 with one or more of its ligands (e.g.,
MAdCAM-1, fibronectin). In a particularly preferred embodiment,
biologically functional fragments can inhibit binding of α4β7
to the mucosal addressin (MAdCAM-1). Examples of biologically functional
antibody fragments which can be administered as described herein include
fragments capable of binding to an α4β7 integrin, such as
single chain antibodies, Fv, Fab, Fab' and F(ab')2 fragments. Such
fragments can be produced by enzymatic cleavage or by recombinant
techniques. For example, papain or pepsin cleavage can generate Fab or
F(ab')2 fragments, respectively. Other proteases with the requisite
substrate specificity can also be used to generate Fab, F(ab')2 or
other antigen-binding fragments. Antibodies can also be produced in a
variety of truncated forms using antibody genes in which one or more stop
codons have been introduced upstream of the natural stop site. For
example, a chimeric gene encoding a F(ab')2 heavy chain portion can
be designed to include DNA sequences encoding the CH1 domain and
hinge region of the heavy chain.

[0021] The term "humanized immunoglobulin" as used herein refers to an
immunoglobulin (antibody) comprising portions of immunoglobulins of
different origin, wherein at least one portion is of human origin. For
example, the humanized antibody can comprise portions derived from an
immunoglobulin of nonhuman origin with the requisite specificity, such as
a mouse, and from immunoglobulin sequences of human origin (e.g.,
chimeric immunoglobulin), joined together chemically by conventional
techniques (e.g., synthetic) or prepared as a contiguous polypeptide
using recombinant DNA technology (e.g., DNA encoding the protein portions
of the chimeric antibody can be expressed to produce a contiguous
polypeptide chain). Another example of a humanized immunoglobulin is an
immunoglobulin containing one or more immunoglobulin chains comprising a
CDR derived from an antibody of nonhuman origin and a framework region
derived from a light and/or heavy chain of human origin (e.g.,
CDR-grafted antibodies with or without framework changes). Chimeric or
CDR-grafted single chain antibodies are also encompassed by the term
humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No.
4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al.,
U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1;
Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European
Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter,
European Patent No. 0,239,400 B 1; Queen et al., European Patent No. 0
451 216 B1; Padlan, E. A. et al., European Patent Application No.
0,519,596 A1. See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston,
U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426
(1988)), regarding single chain antibodies. In particular embodiments,
the humanized immunoglobulin can include an immunoglobulin chain (e.g.,
heavy chain) having a variable region of non-human origin (e.g., murine
origin) and at least a portion of a human constant region (e.g,
Cγ1), and an immunoglobulin chain (e.g., light chain) where at
least one CDR is of non-human origin (e.g., murine origin) and the
framework regions (FR1, FR2, FR3, FR4) and, optionally, the constant
region (e.g., Cκ, Cλ) are of human origin.

[0022] The antigen binding region of the humanized immunoglobulin (the
nonhuman portion) can be derived from an immunoglobulin of nonhuman
origin (referred to as a donor immunoglobulin) having binding specificity
for α4β7 integrin. For example, a suitable antigen binding
region can be derived from the murine Act-1 monoclonal antibody
(Lazarovits, A. I. et al., J. Immunol., 133(4): 1857-1862 (1984)). Other
sources include α4β7 integrin-specific antibodies obtained
from nonhuman sources, such as rodent (e.g., mouse, rat), rabbit, pig
goat or non-human primate (e.g., monkey). Other polyclonal or monoclonal
antibodies, such as antibodies which bind to the same or similar epitope
as the Act-1 antibody, or LDP-02, can be made (e.g., Kohler et al.,
Nature, 256:495-497 (1975); Harlow et al., 1988, Antibodies: A Laboratory
Manual, (Cold Spring Harbor, N.Y.); and Current Protocols in Molecular
Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al., Eds. (John
Wiley & Sons: New York, N.Y.), Chapter 11 (1991)).

[0024] In one embodiment, the antigen binding region of the humanized
immunoglobulin comprises a CDR of nonhuman origin. In this embodiment,
the humanized immunoglobulin having binding specificity for
α4β7 integrin comprises at least one CDR of nonhuman origin.
For example, CDRs can be derived from the light and heavy chain variable
regions of immunoglobulins of nonhuman origin, such that a humanized
immunoglobulin includes substantially heavy chain CDR1, CDR2 and/or CDR3,
and/or light chain CDR1, CDR2 and/or CDR3, from one or more
immunoglobulins of nonhuman origin, and the resulting humanized
immunoglobulin has binding specificity for α4β7 integrin.
Preferably, all three CDRs of a selected chain are substantially the same
as the CDRs of the corresponding chain of a donor, and more preferably,
all six CDRs of the light and heavy chains are substantially the same as
the CDRs of the corresponding donor chains. In a preferred embodiment,
the one or more CDRs of nonhuman origin have the amino acid sequences of
the CDRs of murine Act-1 Ab (SEQ ID Nos. 9-14).

[0026] If present, human framework regions (e.g., of the light chain
variable region) are preferably derived from a human antibody variable
region having sequence similarity to the analogous region (e.g., light
chain variable region) of the antigen binding region donor. Other sources
of framework regions for portions of human origin of a humanized
immunoglobulin include human variable consensus sequences (see e.g.,
Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991); Carter
et al., WO 94/04679, published Mar. 3, 1994)). For example, the sequence
of the antibody or variable region used to obtain the nonhuman portion
can be compared to human sequences as described in Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest. Fifth Edition, U.S.
Department of Health and Human Services, U.S. Government Printing Office
(1991). In a particularly preferred embodiment, the framework regions of
a humanized immunoglobulin chain are derived from a human variable region
having at least about 65% overall sequence identity, and preferably at
least about 70% overall sequence identity, with the variable region of
the nonhuman donor antibody (e.g., mouse Act-1 antibody). A human portion
can also be derived from a human antibody having at least about 65%
sequence identity, and preferably at least about 70% sequence identity,
within the particular portion (e.g., FR) being used, when compared to the
equivalent portion (e.g., FR) of the nonhuman donor. Amino acid sequence
identity can be determined using a suitable sequence alignment algorithm,
such as the Lasergene system (DNASTAR, Inc., Madison, Wis.), using the
default parameters.

[0027] In one embodiment, the humanized immunoglobulin comprises at least
one of the framework regions (FR) derived from one or more chains of an
antibody of human origin. Thus, the FR can include a FR1 and/or FR2
and/or FR3 and/or FR4 derived from one or more antibodies of human
origin. Preferably, the human portion of a selected humanized chain
includes FR1, FR2, FR3 and FR4 derived from a variable region of human
origin (e.g., from a human immunoglobulin chain, from a human consensus
sequence).

[0028] The immunoglobulin portions of nonhuman and human origin for use in
preparing humanized antibodies can have sequences identical to
immunoglobulins or immunoglobulin portions from which they are derived or
to variants thereof. Such variants include mutants differing by the
addition, deletion, or substitution of one or more residues. As indicated
above, the CDRs which are of nonhuman origin are substantially the same
as in the nonhuman donor, and preferably are identical to the CDRs of the
nonhuman donor. Changes in the framework region, such as those which
substitute a residue of the framework region of human origin with a
residue from the corresponding position of the donor, can be made. One or
more mutations in the framework region can be made, including deletions,
insertions and substitutions of one or more amino acids. For a selected
humanized antibody or chain, suitable framework mutations can be
designed. Preferably, the humanized immunoglobulins can bind
α4β7 integrin with an affinity similar to or better than that
of the nonhuman donor. Variants can be produced by a variety of suitable
methods, including mutagenesis of nonhuman donor or acceptor human
chains.

[0029] Immunoglobulins (e.g., human and/or humanized immunoglobulins)
having binding specificity for human α4β7 integrin include
immunoglobulins (including antigen-binding fragments) which can bind
determinants (epitopes) of the α4 chain (e.g., mAb HP1/2 (Pulido,
et al., J Biol Chem 266:10241-10245 (1991), murine MAb 21.6 and humanized
MAb 21.6 (Bendig et al., U.S. Pat. No. 5,840,299)) and/or the β7
chain of the α4β7 heterodimer. For example, in particular
embodiments, the human or humanized immunoglobulin can specifically or
selectively bind a determinant of the α4β7 complex, but not
bind determinants (epitopes) on the α4 chain or the β7 chain.
In one embodiment, the human or humanized immunoglobulin can have binding
specificity for a combinatorial epitope on the α4β7
heterodimer. Such an immunoglobulin can bind α4β7 and not bind
α4β1, for example. Antibodies which have binding specificity
for the α4β7 complex include, murine Act-1 antibody and a
humanized Act-1 referred to as LDP-02 (see, WO 98/06248 by LeukoSite,
Inc., published Feb. 19, 1998 and U.S. application Ser. No. 08/700,737,
filed Aug. 15, 1996, the entire teachings of which are both incorporated
herein by reference). In a preferred embodiment, the humanized
immunoglobulin has at least one function characteristic of murine Act-1
antibody, such as binding function (e.g., having specificity for
α4β7 integrin, having the same or similar epitopic
specificity), and/or inhibitory function (e.g., the ability to inhibit
α4β7-dependent adhesion in vitro and/or in vivo, such as the
ability to inhibit α4β7 integrin binding to MAdCAM-1 in vitro
and/or in vivo, or the ability to inhibit the binding of a cell bearing
α4β7 integrin to a ligand thereof (e.g., a cell bearing
MAdCAM-1)). Thus, preferred humanized immunoglobulins can have the
binding specificity of the murine Act-1 antibody, the epitopic
specificity of murine Act-1 antibody (e.g., can compete with murine
Act-1, a chimeric Act-1 antibody, or humanized Act-1 (e.g., LDP-02) for
binding to α4β7 (e.g., on a cell bearing α4β7
integrin)), and/or inhibitory function. A particularly preferred
humanized Ab for administration in accordance with the method is LDP-02.

[0030] The binding function of a human or humanized immunoglobulin having
binding specificity for α4β7 integrin can be detected by
standard immunological methods, for example using assays which monitor
formation of a complex between humanized immunoglobulin and
α4β7 integrin (e.g., a membrane fraction comprising
α4β7 integrin, on a cell bearing α4β7 integrin,
such as a human lymphocyte (e.g., a lymphocyte of the
CD4+α4hi,β1lo subset), human lymphocyte cell line or
recombinant host cell comprising nucleic acid encoding α4 and/or
β7 which expresses α4β7 integrin). Binding and/or
adhesion assays or other suitable methods can also be used in procedures
for the identification and/or isolation of immunoglobulins (e.g., human
and/or humanized immunoglobulins) (e.g., from a library) with the
requisite specificity (e.g., an assay which monitors adhesion between a
cell bearing an α4β7 integrin and a ligand thereof (e.g., a
second cell expressing MAdCAM, an immobilized MAdCAM fusion protein
(e.g., MAdCAM-Ig chimera)), or other suitable methods.

[0031] The immunoglobulin portions of nonhuman and human origin for use in
preparing humanized immunoglobulins include light chains, heavy chains
and portions of light and heavy chains. These immunoglobulin portions can
be obtained or derived from immunoglobulins (e.g., by de novo synthesis
of a portion), or nucleic acids encoding an immunoglobulin or chain
thereof having the desired property (e.g., binds α4β7
integrin, sequence similarity) can be produced and expressed. Humanized
immunoglobulins comprising the desired portions (e.g., antigen binding
region, CDR, FR, constant region) of human and nonhuman origin can be
produced using synthetic and/or recombinant nucleic acids to prepare
genes (e.g., cDNA) encoding the desired humanized chain. To prepare a
portion of a chain, one or more stop codons can be introduced at the
desired position. For example, nucleic acid (e.g., DNA) sequences coding
for newly designed humanized variable regions can be constructed using
PCR mutagenesis methods to alter existing DNA sequences (see e.g.,
Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989)). PCR primers coding
for the new CDRs can be hybridized to a DNA template of a previously
humanized variable region which is based on the same, or a very similar,
human variable region (Sato, K., et al., Cancer Research 53:851-856
(1993)). If a similar DNA sequence is not available for use as a
template, a nucleic acid comprising a sequence encoding a variable region
sequence can be constructed from synthetic oligonucleotides (see e.g.,
Kolbinger, F., Protein Engineering 8:971-980 (1993)). A sequence encoding
a signal peptide can also be incorporated into the nucleic acid (e.g., on
synthesis, upon insertion into a vector). If the natural signal peptide
sequence is unavailable, a signal peptide sequence from another antibody
can be used (see, e.g., Kettleborough, C. A., Protein Engineering
4:773-783 (1991)). Using these methods, methods described herein or other
suitable methods, variants can be readily produced. In one embodiment,
cloned variable regions (e.g., of LDP-02) can be mutagenized, and
sequences encoding variants with the desired specificity can be selected
(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No.
5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

[0032] Human and/or humanized immunoglobulins can be administered (e.g.,
to a human) for therapeutic and/or diagnostic purposes in accordance with
the method of the invention. For example, an effective amount of a human
and/or humanized immunoglobulins having binding specificity for
α4β7 integrin can be administered to a human to treat a
disease associated with leukocyte infiltration of mucosal tissues (e.g.,
inflammatory bowel disease, such as Crohn's disease or ulcerative
colitis). Treatment includes therapeutic or prophylactic treatment (e.g.,
maintenance therapy). According to the method, the disease can be
prevented or delayed (e.g., delayed onset, prolonged remission or
quiescence) or the severity of disease can be reduced in whole or in
part.

[0033] In one embodiment, no more than about 8 mg of immunoglobulin per kg
body weight is administered during a period of about 1 month. In
additional embodiments, no more than about 7 or about 6 or about 5 or
about 4 or about 3 or about 2 or about 1 mg of immunoglobulin per kg body
weight is administered during a period of about 1 month. As used herein,
the term "month" refers to a calendar month and encompasses periods of
28, 29, 30 and 31 days. When an antigen-binding fragment of a human or
humanized immunoglobulin is to be administered, the amount which is
administered during the period of about one month can be adjusted in
accordance with the size of the fragment. For example, if the
antigen-binding fragment is about half the size of the intact antibody by
weight (e.g., measured in kDa), the amount administered during a period
of about 1 month can be about 4 mg per kg body weight or less. The amount
of immunoglobulin or antigen-binding fragment administered can be
expressed as mg/kg body weight or using any other suitable units. For
example, the amount of immunoglobulin or antigen-binding fragment
administered can be expressed as moles of antigen binding sites per kg
body weight. The number of moles of antigen-binding sites is dependent
upon the size, quantity and valency of the immunoglobulin or fragment and
can be readily determined. For example, IgG and F(ab')2 fragments
thereof are divalent and a dose which comprises 1 nanomole of IgG or
F(ab')2 fragment comprises 2 nanomoles of antigen-binding sites. The
size of an antibody or antigen-binding fragment can be determined using
any suitable method (e.g., gel filtration).

[0034] The human or humanized antibody or antigen-binding fragment can be
administered in a single dose or in an initial dose followed by one or
more subsequent doses. When multiple doses are desired, the interval
between doses and the amount of immunoglobulin or antigen-binding
fragment can be adjusted to achieve the desired therapeutic and/or
diagnostic effect. For example, each of the doses to be administered can
independently comprise up to about 8 mg immunoglobulin or fragment per kg
body weight. When a dose comprises about 8 mg immunoglobulin or fragment
per kg body weight the minimum interval before a subsequent dose is
administered is a period of about 1 month. Preferably, each dose
independently comprises about 0.1 to about 8 mg or about 0.1 to about 5
mg immunoglobulin or fragment per kg body weight. More preferably, each
dose independently comprises about 0.1 to about 2.5 mg immunoglobulin or
fragment per kg body weight. Most preferably, each dose independently
comprises about 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mg
immunoglobulin or fragment per kg body weight.

[0035] The interval between any two doses (e.g., initial dose and first
subsequent dose, first subsequent dose and second subsequent dose) can
independently vary from a few seconds or minutes to about 120 days or
more. For example, the initial dose can be administered and a first
subsequent dose can be administered about 1 day later. Thereafter, second
and third subsequent doses can be administered at intervals of about 1
month. Generally the minimum interval between doses is a period of at
least about 1 day or at least about 7 days. In particular embodiments,
the minimum interval between doses is a period of at least about 14 days,
or at least about 21 days or at least about 1 month (e.g., 28, 29, 30, 31
days). In additional embodiments, the interval between doses can be at
least about 40, about 50, about 60, about 70, about 80, about 90, about
100, about 110 or about 120 days.

[0036] The amount of human or humanized immunjoglobulin or antigen-binding
fragments thereof administered in each dose can be an amount which is
sufficient to produce a desired pharmacokinetic or pharmacodynamic
effect. A variety of pharmacokinetic and pharmacodynamic parameters of
human and/or humanized immunoglobulins or antigen-binding fragments
thereof can be measured using suitable methods. For instance,
pharmacodymanic parameters of antibodies and antigen-binding fragments
(e.g., antigen saturation, antibody-induced inhibition of expression of
antigen) can be measured using a suitable immunoassay. For example, as
described herein, α4β7 signal (i.e., binding of labeled
antibody to α4β7) following administration of LDP-02 was
measured by flow cytometry. The results of the assay revealed that
administration of LDP-02 can result in saturation of α4β7
and/or inhibition of expression of α4β7 on the surface of
circulating lymphocytes.

[0037] Accordingly, each dose to be administered can comprise an amount of
immunoglobulin or fragment which is sufficient to achieve a) about 50% or
greater saturation of α4β7 integrin binding sites on
circulating lymphocytes (e.g., CD8+ cells) and/or b) about 50% or greater
inhibition of α4β7 integrin expression on the cell surface of
circulating lymphocytes for a period of at least about 10 days following
administration of the dose. In other embodiments, each dose can comprise
an amount of immunoglobulin or fragment which is sufficient to achieve
and maintain a) about 60% or greater, about 70% or greater, about 80% or
greater or about 85% or greater saturation of α4β7 integrin
binding sites on circulating lymphocytes and/or b) about 60% or greater,
about 70% or greater, about 80% or greater or about 85% or greater
inhibition of α4β7 integrin expression on the cell surface of
circulating lymphocytes for a period of at least about 10 days following
administration of the dose.

[0038] In other particular embodiments, each dose can comprise an amount
of immunoglobulin or fragment which is sufficient to achieve a desired
degree of saturation of α4β7 integrin binding sites on
circulating lymphocytes (e.g., CD8+ cells) and/or inhibit expression of
α4β7 integrin on the cell surface of circulating lymphocytes
to the desired degree for a period of at least about 14 days, at least
about 20 days, at least about 25 days or at least about one month
following administration of the dose. In additional embodiments, each
dose can comprise an amount of immunoglobulin or fragment which is
sufficient to achieve a desired degree of saturation of α4β7
integrin binding sites on circulating lymphocytes (e.g., CD8+ cells)
and/or inhibit expression of α4β7 integrin on the cell surface
of circulating lymphocytes to the desired degree for a period of at least
about 40, about 50, about 60, about 70, about 80, about 90, about 100,
about 110 or about 120 days.

[0039] Suitable assays for determining the dose of antibody required to
achieve a desired serum concentration or to saturate and/or inhibit
expression of a target antigen can be readily designed. For example, a
flow cytometry based assay can be used to measure α4β7
expression on the surface of cells isolated from a subject following
administration of an immunoglobulin (e.g., human, humanized) which binds
to α4β7. In one embodiment, a murine antibody which binds
human α4β7 can be used. Preferably the murine antibody can
bind to an epitope on α4β7 which is distinct from the epitope
bound by the human or humanized immunoglobulin and the binding of the
murine antibody to α4β7 is not inhibited (e.g., blocked) by
the prior binding of the humanized immunoglobulin. Murine antibodies or
other antibodies with these properties can be prepared and selected using
the methods described herein or other suitable methods. The level of
α4β7 expression on circulating lymphocytes (e.g., CD8+ cells)
isolated from a human can be measured or determined using each of the
antibodies (i.e., immunoglobulin to be administered, murine antibody) by
flow cytometry or other suitable methods. Then, the humanized antibody
can be administered to the human, peripheral blood can be drawn at
predetermined times following the administration and lymphocytes can be
isolated (e.g., by density gradient centrifugation) for analysis. The
peripheral blood lymphocytes (e.g., CD8+ cells) can be stained with each
of the antibodies and the amount of α4β7 detected by each
antibody can be measured or detected by flow cytometry or other suitable
methods. A decrease in the amount of α4β7 integrin measured or
determined using the human or humanized immunoglobulin is indicative of
a) persistent integrin occupancy by the immunoglobulin (e.g., antigen
saturation) and/or b) inhibition of α4β7 expression on the
surface of the lymphocytes (e.g., down modulation of α4β7,
shedding of α4β7). A decrease in the amount of α4β7
integrin measured or detected using the human or humanized immunoglobulin
together with no change in the amount of α4β7 integrin
measured or determined using the murine antibody is indicative of
persistent occupancy of α4β7 (e.g., saturation) by the
humanized immunoglobulin. A decrease in the amount of α4β7
integrin measured or detected using the human or humanized immunoglobulin
together with a decrease in the amount of α4β7 integrin
measured or detected using the murine antibody is indicative of
inhibition of α4β7 expression on the surface of circulating
lymphocytes.

[0040] Pharmacokinetic parameters, such as the serum concentration of
antibody over time following administration of said antibody can be
measured using an immunoassay such as an ELISA or cell-based assay. For
example, as described herein, the serum concentration of a humanized
anti-α4β7 immunoglobulin (LDP-02) at predetermined time points
following a single administration of antibody (LDP-02) was measured using
a cell-based assay. The results of the assay revealed that the serum
concentration of LDP-02 can remain elevated (e.g., at or above 1
μg/ml) for a period of about 10 days or more following administration
of the humanized antibody. The prolonged presence of LDP-02 in the serum
can be indicative of superior efficacy as a result of persistent
inhibition of α4β7 function, for example persistent inhibition
of α4β7 mediated adhesion of leukocytes to MAdCAM.

[0041] Accordingly, each dose to be administered can comprise an amount of
immunoglobulin or fragment which is sufficient to achieve and maintain a
serum concentration of at least about 1 μg/mL for a period of at least
about 10 days following administration of the dose. In particular
embodiments, each dose can comprise amount of immunoglobulin or fragment
which is sufficient to achieve and maintain a serum concentration of at
least about 1 μg/mL for a period of at least about 14 days, at least
about 20 days, at least about 25 days or at least about one month
following administration of the dose. In additional embodiments, each
dose can comprise amount of immunoglobulin or fragment which is
sufficient to achieve and maintain a serum concentration of at least
about 1 μg/mL for a period of at least about 40, about 50, about 60,
about 70, about 80, about 90, about 100, about 110 or about 120 days.

[0042] As discussed herein, antigen-binding fragments of a human or
humanized immunoglobulin can be substantially smaller and, therefore,
bind more antigen (α4β7) per unit of protein (μg) than
intact or native immunoglobulin. Accordingly, the serum concentration of
an antigen-binding fragment of a human or humanized immunoglobulin which
can be indicative of superior efficacy can be lower than 1 μg/mL.
Thus, when administration of an antigen-binding fragment of a human or
humanized immunoglobulin is desired, the dose can comprise an amount of
antigen-binding fragment which is sufficient to achieve a serum
concentration which is proportionate to 1 μg/mL for an intact
immunoglobulin. For example, if the antigen-binding fragment is about
half the size of the intact antibody by weight (e.g., measured in kDa),
the dose can comprise an amount sufficient to achieve and maintain a
serum concentration of about 0.5 μg/mL for a period of at least about
10 days. The desired serum concentration of immunoglobulin or
antigen-binding fragment can be expressed as μg/mL or using any other
suitable units. For example, the amount of immunoglobulin or
antigen-binding fragment administered can be expressed as moles of
antigen binding sites per volume of serum (e.g., M). Human and humanized
immunoglobulins can be administered in accordance with the present
invention for in vivo diagnostic applications or to modulate
α4β7 integrin function in therapeutic (including prophylactic)
applications. For example, human and humanized immunoglobulins can be
used to detect and/or measure the level of an α4β7 integrin in
a subject. For example, a humanized immunoglobulin having binding
specificity for α4β7 integrin can be administered to a human
and antibody-α4β7 integrin complexes which are formed can be
detected using suitable methods. For example, the humanized antibody can
be labeled with, for example, radionuclides (125I, 111In,
technetium-99m), an epitope label (tag), an affinity label (e.g., biotin,
avidin), a spin label, an enzyme, a fluorescent group or a
chemiluminescent group and suitable detection methods can be used. In an
application of the method, humanized immunoglobulins can be used to
analyze normal versus inflamed tissues (e.g., from a human) for
α4β7 integrin reactivity and/or expression (e.g.
radiologically) or to detect associations between IBD or other conditions
and increased expression of α4β7 (e.g., in affected tissues).
The immunoglobulins described herein can be administered in accordance
with the method of the invention for assessment of the presence of
α4β7 integrin in normal versus inflamed tissues, through which
the presence of disease, disease progress and/or the efficacy of
anti-α4β7 integrin therapy in inflammatory disease can be
assessed.

[0043] Human and humanized immunoglobulins (including antigen-binding
fragments) can be administered to an individual to modulate (e.g.,
inhibit (reduce or prevent)) binding function and/or leukocyte (e.g.,
lymphocyte, monocyte) infiltration function of α4β7 integrin.
For example, human and humanized immunoglobulins which inhibit the
binding of α4β7 integrin to a ligand (i.e., one or more
ligands) can be administered according to the method for the treatment of
diseases associated with leukocyte (e.g., lymphocyte, monocyte)
infiltration of tissues (including recruitment and/or accumulation of
leukocytes in tissues), particularly of tissues which express the
molecule MAdCAM. An effective amount of a human immunoglobulin or
antigen-binding fragment thereof, or humanized immunoglobulin or
antigen-binding fragment thereof (i.e., one or more immunoglobulins or
fragments) is administered to an individual (e.g., a mammal, such as a
human or other primate) in order to treat such a disease. For example,
inflammatory diseases, including diseases which are associated with
leukocyte infiltration of the gastrointestinal tract (including
gut-associated endothelium), other mucosal tissues, or tissues expressing
the molecule MAdCAM-1 (e.g., gut-associated tissues, such as venules of
the lamina propria of the small and large intestine; and mammary gland
(e.g., lactating mammary gland)), can be treated according to the present
method. Similarly, an individual having a disease associated with
leukocyte infiltration of tissues as a result of binding of leukocytes to
cells (e.g., endothelial cells) expressing MAdCAM-1 can be treated
according to the present invention.

[0045] Pancreatitis and insulin-dependent diabetes mellitus are other
diseases which can be treated using the present method. It has been
reported that MAdCAM-1 is expressed by some vessels in the exocrine
pancreas from NOD (nonobese diabetic) mice, as well as from BALB/c and
SJL mice. Expression of MAdCAM-1 was reportedly induced on endothelium in
inflamed islets of the pancreas of the NOD mouse, and MAdCAM-1 was the
predominant addressin expressed by NOD islet endothelium at early stages
of insulitis (Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515
(1993)). Further, accumulation of lymphocytes expressing α4β7
within islets was observed, and MAdCAM-1 was implicated in the binding of
lymphoma cells via α4β7 to vessels from inflamed islets
(Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515 (1993)).

[0046] Examples of inflammatory diseases associated with mucosal tissues
which can be treated according to the present method include mastitis
(mammary gland), cholecystitis, cholangitis or pericholangitis (bile duct
and surrounding tissue of the liver), chronic bronchitis, chronic
sinusitis, asthma, and graft versus host disease (e.g., in the
gastrointestinal tract). As seen in Crohn's disease, inflammation often
extends beyond the mucosal surface, accordingly chronic inflammatory
diseases of the lung which result in interstitial fibrosis, such as
hypersensitivity pneumonitis, collagen diseases, sarcoidosis, and other
idiopathic conditions can be amenable to treatment.

[0047] Treatment can be curative, induce remission or quiescence or
prevent relapse or recurrence of active disease. According to the method,
treatment can be episodic or chronic (e.g., chronic treatment of active
disease, to maintain quiescent disease, to induce quiescence and maintain
quiescence), for example.

[0048] In a particularly preferred embodiment, a human or humanized
immunoglobulin having binding specificity for α4β7 integrin is
administered to a human having inflammatory bowel disease, such as
ulcerative colitis or Crohn's disease. The immunoglobulin can be
administered to treat active disease and/or to maintain quiescence (i.e.,
inhibit relapse or recurrence). In a particular embodiment, the human or
humanized immunoglobulin can be administered to maintain quiescence of
inflammatory bowel disease which has been induced by treatment with one
or more other agents (e.g., steroids (prednisone, prednisolone,
adrenocorticotrophic hormone (ACTH)), cyclosporin A, FK506, antibody
having binding specificity for TNFα (infliximab, CDP571),
azathioprene, 6-mercaptopurine, 5-aminosalicylic acid (5-ASA) or
compounds containing 5-ASA (e.g., sulfsalazine, olsalazine, balsalazide)
antibiotics (e.g., metronidazole), interleukins (IL-10, IL-11), nicotine,
heparin, thalidomide, lidocane) or surgery (e.g., intestinal resection).

[0049] The human immunoglobulin or antigen-binding fragment thereof, or
humanized immunoglobulin or antigen-binding fragment thereof is
administered in an effective amount. For therapy, an effective amount is
an amount sufficient to achieve the desired therapeutic (including
prophylactic) effect (such as an amount sufficient to reduce or prevent
α4β7 integrin-mediated binding to a ligand thereof and/or
signalling, thereby inhibiting leukocyte adhesion and infiltration and/or
associated cellular responses in an amount sufficient to induce remission
or prevent relapse or recurrence of disease). The human immunoglobulin or
antigen-binding fragment thereof, or humanized immunoglobulin or
antigen-binding fragment thereof can be administered in a single dose or
in an initial dose followed by one or more subsequent doses as described
herein. The amount of immunoglobulin or antigen-binding fragment
administered in a particular dose as well as the interval between doses
can depend on the characteristics of the individual, such as general
health, age, sex, body weight and tolerance to drugs as well as the type
and severity of disease. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors.

[0050] According to the method, the human or humanized immunoglobulin can
be administered to an individual (e.g., a human) alone or in conjunction
with another agent (i.e., one or more additional agents). A human or
humanized immunoglobulin can be administered before, along with or
subsequent to administration of the additional agent. In one embodiment,
more than one human or humanized immunoglobulin which inhibits the
binding of α4β7 integrin to its ligands is administered. In
another embodiment, an antibody (e.g, human antibody, humanized
antibody), such as an anti-MAdCAM-1, anti-VCAM-1, or anti-ICAM-1
antibody, which inhibits the binding of leukocytes to an endothelial
ligand is administered in addition to a human or humanized immunoglobulin
which binds α4β7 integrin. In yet another embodiment, an
additional pharmacologically active ingredient (e.g., an antiinflammatory
compound, such as 5-aminosalicylic acid (5-ASA) or compounds containing
5-ASA (e.g., sulfsalazine, olsalazine, balsalazide), another
non-steroidal antiinflammatory compound, or a steroidal antiinflammatory
compound (e.g., prednisone, prednisolone, adrenocorticotrophic hormone
(ACTH)), immunosuppressive agents (azathioprene, 6-mercaptopurine,
cyclosporin A, FK506), immunomodulators (e.g., antibody having binding
specificity for TNFα (infliximab, CDP571), thalidomide,
interleukins (e.g., recombinant human IL-10, recombinant human IL-11)),
antibiotics (e.g., metronidazole), nicotine, heparin, lidocaine) can be
administered in conjunction with a humanized immunoglobulin of the
present invention.

[0052] The human immunoglobulin or antigen-binding fragment thereof and/or
the humanized immunoglobulin or antigen-binding fragment thereof can be
administered to the individual as part of a pharmaceutical or
physiological composition for the treatment of a disease associated with
leukocyte infiltration of mucosal tissues (e.g., inflammatory bowel
disease (e.g., ulcerative colitis, Crohn's disease). Such a composition
can comprise an immunoglobulin or antigen-binding fragment having binding
specificity for α4β7 integrin as described herein, and a
pharmaceutically or physiologically acceptable carrier. Pharmaceutical or
physiological compositions for co-therapy can comprise an immunoglobulin
or antigen-binding fragment having binding specificity for
α4β7 integrin and one or more additional therapeutic agents.
An immunoglobulin or antigen-binding fragment having binding specificity
for α4β7 integrin function and an additional therapeutic agent
can be components of separate compositions which can be mixed together
prior to administration or administered separately. Formulation will vary
according to the route of administration selected (e.g., solution,
emulsion, capsule). Suitable carriers can contain inert ingredients which
do not interact with the immunoglobulin or antigen-binding fragment
and/or additional therapeutic agent. Standard pharmaceutical formulation
techniques can be employed, such as those described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable
carriers for parenteral administration include, for example, sterile
water, physiological saline, bacteriostatic saline (saline containing
about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's
solution, Ringer's-lactate and the like. Methods for encapsulating
compositions (such as in a coating of hard gelatin or cyclodextran) are
known in the art (Baker, et al., "Controlled Release of Biological Active
Agents", John Wiley and Sons, 1986). For inhalation, the agent can be
solubilized and loaded into a suitable dispenser for administration
(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

[0053] The present invention will now be illustrated by the following
Examples, which are not intended to be limiting in any way,

EXAMPLES

Introduction

[0054] LDP-02 is a humanized IgG1 monoclonal antibody that binds
α4β7 integrin, a cell surface glycoprotein present on the
surface of most T and B lymphocytes. α4β7 mediates lymphocyte
trafficking to gastrointestinal mucosa and gut-associated lymphoid tissue
through adhesion interaction with the homing receptor MAdCAM-1. By
blocking α4β7-MAdCAM-1 interactions, LDP-02 can inhibit the
recruitment of leukocytes from the vasculature to the gastrointestinal
mucosa, thus having a beneficial effect on the inflammatory activity in
patients afflicted with inflammatory bowel disease (IBD) such as
ulcerative colitis and Crohn's Disease.

[0055] This section presents information from the two LDP-02 clinical
trials that have been completed. These trials include one completed Phase
I study conducted in healthy subjects (Study L297-007) and one completed
Phase Ib/IIa trials in patients with ulcerative colitis (UC) (Study
L297-006). Table 1 describes each of the studies

[0056] Study L297-007 entitled, "A Placebo-Controlled, Double-Blind,
Rising Dose Study Investigating the Tolerability, Pharmacodynamics and
Pharmacokinetics of LDP-02 Given by the Subcutaneous and Intravenous
Routes in Healthy Male Volunteers" has been completed and final results
are presented in this section.

Study Design

[0057] Study L297-007 was a randomized, double-blind, placebo-controlled,
ascending single-dose study in healthy male volunteers, Healthy male
volunteers 18 to 50 years of age meeting all inclusion/exclusion criteria
were enrolled in the study sequentially by study group and, within each
study group, were randomly assigned to receive LDP-02 or placebo (i.e.,
isotonic sodium citrate buffer). To minimize risk to subjects, safety and
tolerability were reviewed at each dose level prior to escalating to the
next dose level. The treatment groups and numbers of subjects planned for
the study are shown in Table 2.

[0058] On study Day 1, LDP-02 or placebo was administered either SC into
the thigh (Group 1 SC dosing only) or via a 30 minute constant rate IV
infusion (Groups 1-4). Safety assessments included recording of adverse
events, physical examinations, vital signs, clinical laboratories (i.e.,
hematology, blood chemistries, and urinalysis), plasma cytokine levels,
and 12-lead electrocardiograms (ECGs). In addition, since this was the
first clinical trial of LDP-02, continuous cardiac monitoring was carried
out pre-dose through 4 hours post-dose. Blood samples were obtained to
assess anti-antibody response to LDP-02, cytokine levels, serum LDP-02
concentration (pharmacokinetics), and saturation and binding site
occupation of α4β7 receptors and lymphocyte subsets
(pharmacodynamics). Study assessments were conducted at specified times
through 36 days post-treatment. Following the results of the Day 36
pharmacokinetic and pharmacodynamic (immunological) analyses, the
protocol was amended to allow additional blood draws for subjects who
received LDP-02. These blood draws were used to follow LDP-02 serum
levels until they became non-quantifiable (i.e., below the limit of
quantification [BLQ]) and to ensure that α4β7 saturation and
memory cell populations had returned to baseline (pre-dose) levels. This
amendment was particularly important in the higher dose groups where the
characteristics of terminal phase kinetics were not well established by
Day 36.

Study Results

Pharmacokinetics

[0059] The assay of LDP-02 in serum was performed using a validated
cell-based assay. Standards and samples were incubated with a target cell
line (HUT-78) which expresses the α4β7 antigen. After washing,
a fluorescently labeled polyclonal anti-human IgG1 was added.
Fluorescence intensity was measured by flow cytometry and compared with
the fluorescence intensity of LDP-02 standards. The effective serum
concentration of LDP-02 was then defined by comparison of the sample with
a standard curve generated with known concentrations of LDP-02.

[0060] Blood samples for determination of LDP-02 serum concentration were
collected pre-dose, 1, 1.5, 3, 8, 12 and 24 hours after dosing, and on
Days 3, 5, 7, 8, 15, 22, and 36. When it became known that LDP-02 was
still detectable at Day 36, blood draws for subjects who received LDP-02
continued until levels had fallen to below the limits of quantitation of
the assay. Thirteen of the 14 subjects who received LDP-02 returned for
follow-up blood draws up to a maximum of 226 days post-dose.

[0061] LDP-02 concentrations over time by individual patient and mean
pharmacokinetic parameters by LDP-02 dose group are presented in the
Appendix to Study L297-007. Mean LDP-02 serum concentrations over time
are plotted out to the last blood draw for all treatment groups in FIG.
6.

[0062] Values were obtained for the mean single dose IV pharmacokinetic
parameters for the 4 dose groups (Cmax', t1/2z and AUC).
Follow-up samples (i.e., those taken beyond Day 36), where the focus was
on safety, allowed some further characterization of the
concentration-time profiles. The difference in the t1/2z values
between the 2 lower dose groups (0.15 and 0.5 mg/kg) and the higher dose
groups (1.5 and 2.5 mg/kg) of around 10 days could be explained in that
the "true" terminal phase for the higher dose groups had not been
characterized. The non-compartmental pharmacokinetics of the lower doses
of LDP-02 (0.15 and 0.5 mg/kg) were well characterized and non-linear
pharmacokinetics became evident as the dose was increased up to 2.5
mg/kg,

Assessment of the Pharmacodynamic Effect of LDP-02

[0063] Fluorescent activated cell scanning (FACS) analysis was used to
measure the presence of α4β7 sites on peripheral blood
lymphocytes pre- and post-LDP-02 administration. To detect
α4β7 that were recognized by antibody, biotin labeled ACT-1,
the marine homologue of LDP-02, was added to samples of patient blood and
detected using PE-streptavidin. The standardized mean equivalent soluble
fluorescence (MESF) is proportional to the number of detectable
α4β7 sites.

[0064] Serum α4β7 binding over time (MESF values and percentage
of baseline at each post-dose time point) are presented by individual
subject and by treatment group in the Appendix to Study L297-007.

[0065] As measured by FACS analysis, mean saturation of α4β7
integrin on lymphocytes over time (i.e., to Day 36) for each treatment
are presented in FIG. 7.

[0066] As seen in FIG. 7, there was no detection of free α4β7
binding sites on lymphocytes for at least two weeks following
administration of all LDP-02 doses. Between about day 7 and day 22,
α4β7 signal started to return to baseline for the 0.15 mg/kg
IV dose group and for the 0.15 mg/kg SC dose group. Between day 22 and
day 36, α4β7 signal started to return to baseline for the 0.5
mg/kg IV dose group. At the higher doses of LDP-02 studied (1.5, and 2.5
mg/kg) loss of α4β7 signal persisted for longer than 36 days
following single IV doses. For the 2.5 mg/kg dose group, α4β7
binding saturation continued up to Day 70 (see, data in Appendix to Study
L297-007).

[0067] Follow-up blood sampling up to about Study Day 200 was done to
confirm that free α4β7 binding sites on lymphocytes has
returned to baseline (pre-dose) levels. The initial reappearance of free
α4β7 sites appeared to occur when LDP-02 blood concentrations
became non-detectable.

Conclusions

[0068] The administration of LDP-02 at IV doses of 0.15, 0.50, 1.50, and
2.5 mg/kg and a SC dose of 0.15 mg/kg to healthy male subjects was
well-tolerated.

[0069] Following administration of all LDP-02 doses there was no detection
of free α4β7 binding sites on lymphocytes for approximately
two weeks post-dose. Saturation of α4β7 binding sites
continued for up to approximately 2 weeks post-dosing for the 0.15 mg/kg
IV group and for up to approximately 3 weeks post-dosing for the 0.15
mg/kg SC and 0.5 mg/kg IV groups. Duration of effect persisted for a
month or longer with the 1.5 mg/kg IV dose and continued to approximately
Day 70 with 2.5 mg/kg LDP-02 IV. Follow-up samples obtained after Day 36
demonstrated that expression of free α4β7 binding sites had
returned to baseline (pre-dose levels). No anti-idiotype antibodies were
raised to LDP-02 indicating that it did not initiate a humoral
immunogenic response. The non-compartmental pharmacokinetics of the lower
doses of LDP-02 (0.15 and 0.5 mg/kg) became evident as the dose was
increased up to 2.5 mg/kg.

Appendex to Study L297-007

[0070] LDP-02 Serum Concentration Over Time by Subject by Treatment Group.
Data from individual patients are presented in Tables 4-9.

TABLE-US-00009
TABLE 9
placebo group
Time Time Subject Subject Subject Subject Subject
(hr) (day) # 1 # 7 # 11 # 14 # 17
Pre-Dose Pre-Dose Its Its Its Its Its
1.0 0.042 Its Its Its Its Its
1.5 0.063 Its Its Its Its Its
3.0 0.125 Its Its Its Its Its
8.0 0.333 Its Its Its Its Its
12.0 0.500 Its Its Its Its Its
24.0 1.000 Its Its Its Its Its
72.0 3.000 Its Its Its Its Its
120.0 5.000 Its Its Its Its Its
168.0 7.000 Its Its Its Its Its
192.0 8.000 Its Its Its Its Its
360.0 15.000 Its Its Its Its Its
528.0 22.000 Its Its Its Its Its
864.0 36.000 Its Its Its Its Its
Its = below the limit of detection

L297-007: Serum α4β7 Binding Over Time by Subject by Treatment
Group. Data from individual patients are presented in Tables 15-20. For
each subject the time of blood sampling, MESF of the sample and % of
baseline (pre-dose) MESF is presented.

[0071] The study entitled, "A Single Dose Phase Placebo Controlled,
Randomized, Double-Blind Study to Determine the Safety, Tolerability,
Pharmacokinetics, Pharmacodynamics, and Effectiveness of LDP-02 in
Patients with Moderately Severe Ulcerative Colitis" was completed and
final certain results are presented in this section.

Study Rationale

[0072] Results from the Phase I trial (Example 1. Study L297-007) in
healthy volunteers showed LDP-02 at doses of 0.15 mg/kg SC and IV, 0.5
mg/kg IV, 1.5 mg/kg IV, and 2.5 mg/kg IV was safe and well-tolerated. In
addition, doses of 0.15 mg/kg IV or SC and 0.5 mg/kg IV were shown to
have a t1/2 of approximately 100 to 130 hours and flow cytometry
data showed that unbound α4β7 begins to reappear in the 0.15
mg/kg dosage groups approximately two weeks after dosing. Based upon
these data, LDP-02 dosages of 0.15 mg/kg SC, 0.15 mg IV, 0.5 mg/kg IV,
and 2.0 mg/kg IV were selected for use in the initial study in patients
with ulcerative colitis. This study was designed so that each dose of
LDP-02 was determined to be safe and well-tolerated prior to escalation
to the next dose level.

Study Design

[0073] The study was a randomized, double-blind, placebo-controlled,
ascending single-dose study in patients diagnosed with moderately-severe
ulcerative colitis. Patients with a documented diagnosis of ulcerative
colitis with a minimum disease extent of 25 cm from the anal verge were
potentially eligible for the study. Patients with severe ulcerative
colitis as defined by Truelove-Witts criteria (Br Med J; 2: 1042-1048
(1955)) were excluded. Ulcerative colitis patients who met all
inclusion/exclusion criteria were enrolled sequentially into four study
groups and, within each study group, were randomly assigned to receive
LDP-02 or placebo (i.e., 0.9% sodium chloride). Treatment groups and
numbers of patients enrolled are shown in Table 21.

[0074] Study medication (LDP-02 or placebo) was administered on Day 1
either SC into the thigh or via a 30 minute W infusion. Safety
assessments included recording of adverse events, physical examinations,
vital signs, clinical laboratories (i.e., hematology, blood chemistries,
and urinalysis), plasma cytokine levels, and ECGs. Blood was drawn at
various time points to measure LDP-02 serum concentrations and to assess
the effectiveness of LDP-02 to saturate and block α4β7 binding
receptors on peripheral blood lymphocytes. The effectiveness of LDP-02 to
reduce inflammation in the colon was measured by clinical disease
observations, endoscopic appearance, histopathology, and
immunohistochemistry.

Study Results

[0075] LDP-02. Once the laboratory results were obtained, the patient was
treated with antibiotics and replaced by another patient. There were no
other patients discontinued from the study. As patients were recruited
into the study over time, there was no attempt to balance the treatment
groups with regard to baseline ulcerative colitis history. As such,
severity and duration of ulcerative colitis disease and prior medications
for ulcerative colitis varied from patient to patient and from treatment
group to treatment group. These data are presented in Table 22.

[0076] Although this was primarily a dose-ranging safety and
pharmacokinetics study, various parameters were measured to assess
effectiveness of treatment. Effectiveness assessments included recording
changes from baseline using a modified Baron's (endoscopy) Scoring
System, the Mayo Clinic Disease Activity Index Score, the Powell-Tuck
Disease Activity Index Score, stool frequency, and the Inflammatory Bowel
Disease Questionnaire. Changes from baseline to Day 30 for these
parameters are shown in Table 23. For patients in which there was no Day
30 evaluation, the last post-baseline observation obtained was carried
forward to Day 30.

[0077] As seen from the results presented in Table 23, there was
variability in response among the different treatment groups. The
patients receiving 0.5 mg/kg IV appeared to have the best responses; the
median endoscopic severity score was reduced by two grades and the Mayo
Clinic score was reduced by 10 points with a decrease in stool frequency.
Three of the five patients receiving 0.5 mg/kg IV had a two point
improvement in the modified Baron sigmoidoscopy score which is considered
an endoscopic response; only one patient (compared with a total of five
treated per group) in both the 2.0 mg/kg IV and 0.15 mg/kg SC groups had
an endoscopic response. The placebo group also experienced an improvement
in sigmoidoscopic score and Mayo Clinic score, although both were less in
magnitude when compared to the 0.5 mg/kg IV group. Two of the eight
patients experienced an endoscopic response.

[0078] The number of patients with a complete remission, defined as a zero
on the modified Baron sigmoidoscopic score and on the Mayo Clinic score
at Day 30, are reported in Table 24.

[0079] None of the patients in the placebo group experienced a complete
remission while two patients among those receiving LDP-02 had complete
remissions. The two patients both were in the same group; both patients
received a single administration of 0.5 mg/kg of LDP-02. One of the
patients was receiving concurrent mesalamine therapy, while the other was
receiving concurrent low dose corticosteroid (20 mg prednisone per day
orally).

Pharmacokinetics

[0080] The assay of LDP-02 in serum was performed by Cytometry Associates,
Inc. as previously described (Study L297-007). Blood samples were
collected prior to and immediately following the completion of infusion
(Day 1) and on Days 2, 3, 5, 10, 14, 21, 30 and 60 to assess the
pharmacokinetic profile of LDP-02.

[0081] LDP-02 concentrations over time by individual patient and mean
pharmacokinetic parameters by LDP-02 dose are presented in the Appendix
to study L296-006.

[0082] As seen in FIG. 8, serum levels of LDP-02 for the 0.15 mg/kg IV and
SC groups fall to <1.0 μg/mL to approximately 20 days post-dose.
For the 2.0 mg/kg dose group, LDP-02 levels remain elevated out to
approximately Day 60. Table 25 presents the key pharmacokinetic
parameters by treatment group.

[0083] There does appear to be linearity with dose for the maximum
concentration of LDP-02 and the area under the curve measured after IV
administration. The clearance and the terminal elimination half life
appear to be independent of IV dose administered. The volume of
distribution appears to decrease slightly with increasing doses of IV
LDP-02.

Assessment of the Pharmacodynamic Effect of LDP-02

[0084] FACS analysis to measure the presence of α4β7 sites on
blood lymphocytes was previously described (Study L296-007). Serum
α4β7 binding over time (i.e., MESF values and percentage of
baseline at each post-dose time point) are presented by individual
patient and by treatment group in the Appendix to Study L297-006.

[0085] Mean percent of baseline MESF over time for all treatments are
presented in FIG. 9. As seen in FIG. 9, percent of baseline MESF rapidly
falls to approximately 10% after SC and IV administration of LDP-02 with
duration of effect dependent upon dose. Starting at about day 10,
α4β7 signal started to return to baseline for the 0.15 mg/kg
IV and SC dose groups. However, α4β7 signal started to return
to baseline between day 30 and day 60 for the 0.5 mg/kg IV and 2.0 mg/kg
dose groups.

[0087] The pharmacokinetic and pharmacodynamic data from patients with
ulcerative colitis showed results were consistent with those found in
healthy volunteers. There appeared to be linearity with dose for the
maximum concentration of LDP-02 and area under the curve measured after
IV administration. The clearance and the terminal elimination half life
appeared to be independent of IV dose administration. The volume of
distribution appeared to decrease slightly with increasing doses of IV
LDP-02. The percent of baseline MESF declines to -10% rapidly after SC
and IV administration of LDP-02 with duration of effect dependent upon
dose. For the 0.15 mg/kg IV and SC dose groups, percent of baseline MESF
started returning to baseline approximately 10 days after dosing whereas
this started to occur at -30 days and -60 days for the 0.5 mg/kg IV and
2.0 mg/kg dose groups, respectively.

Appendix to Study L297-006

[0088] LDP-02 Serum Concentration Over Time by Subject by Treatment
Group. Data obtained from individual subjects are presented in Tables
26-30. The data presented in Tables 26-30 are in μg/mL.

Serum α4β7 Binding Over Time by Subject by Treatment Group.
Data obtained from individual subjects are presented in Tables 35-40. For
each subject the time of blood sampling, MESF of the sample and % of
baseline (pre-dose) MESF is presented.

[0090] While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details may be
made therein without departing from the scope of the invention
encompassed by the appended claims.